This invention is directed to methods for detecting oligonucleotides in samples of bodily fluids and/or extracts. Included in the invention are highly sensitive processes and methods which combine oligonucleotide capture techniques, structure-specific enzymes which recognize specific DNA/RNA configurational motifs and detection/labeling systems. These processes and methods can be used, for example, to detect, localize and quantify administered oligonucleotides in bodily fluids and extracts taken from patients undergoing antisense oligonucleotide therapy. Further uses for this invention are for studying the pharmacokinetic properties of oligonucleotides in animal models.
Detection of specific nucleic acid sequences present in a cell, group of cells, or in solution is generally known in the art. Southern (J.Mol.Biol. 98:503-527 (1975)) teaches detection of specific sequences among DNA fragments separated by gel electrophoresis using xe2x80x9cblottingxe2x80x9d or transfer of the DNA fragments to a membrane, followed by hybridization of denatured DNA fragments with radioactive probes and autoradiography. This procedure has been extended to the detection of RNA molecules extracted from cells or tissues. Further improvements have involved faster and more quantitative xe2x80x9cdot-blottingxe2x80x9d procedures to detect DNA or RNA from tissues or cells.
Various methods are known in the art which may be used to detect and characterize specific nucleic acid sequences and sequence changes. These methods must be able to create detectable signals from a very low copy number of the sequence of interest. Example approaches for detecting nucleic acids are; capillary gel electrophoresis (CGE), as described by Cohen et al. U.S. Pat. No. 5,420,265, signal amplification technology, such as polymerase chain reaction (U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis and Mullis et al.) or ligase chain reaction (described by Barany, Proc.Natl.Acad.Sci., 88:189 (1991)), and direct detection technology, such as Southern and Northern Blotting.
Recently, considerable interest has been generated in the development of synthetic oligonucleotides as therapeutic agents. These antisense molecules and approaches to using them have been reviewed in Agarwal, Trends in Biotechnology 10:152-158 (1991). For an antisense therapeutic to be effective, the oligonucleotide must be introduced/administered to a patient and must reach the specific nucleic acid target for which it was designed. Consequently, there is a need to be able to detect oligonucleotide drugs in bodily fluids and extracts. In animal models, radio-labeled oligonucleotides have been administered to the subject and the distribution of the oligonucleotides within the body has been assessed by extraction of the oligonucleotides followed by autoradiography (Agarwal et al., Proc. Natl. Acad. Sci. 88:7595-7599 (1991)). A common aspect of current procedures is the detection of large DNA or RNA molecules ( greater than 100 bp). Due to the small size (20-30 bp) of oligonucleotides used for antisense therapeutics special problems relating to their detection exist, such as for example nonspecific binding or the absence of binding to probes producing false negatives/positives.
Lyamichev et al., Nature Biotechnology 17:292-296 (1999) describe an approach using enzymes responsible for removing the unpaired segments of DNA that arise during DNA synthesis, wherein at the 3xe2x80x2 end of a growing upstream oligonucleotide the sequence at the 5xe2x80x2 end of a downstream oligonucleotide is displaced. In this approach, enzymes, such as eubacterial Pol A DNA polymerases, 5xe2x80x2 to 3xe2x80x2 exonuclease from calf, the 5xe2x80x2 nucleases associated with bacteriophage T5, FEN1, RAD2, and xeroderma pigmentosum-complementation group G endonuclease homologs from eukaryotes, are used to remove the cleavable flaps (redundant non-hybridized single stranded nucleic acid portion on the 5xe2x80x2 end of the downstream segment) created by introducing an upstream oligonucleotide, getting incomplete hybridization, and not extending the primer. Lyamichev et al., describe detection of DNA targets in complex mixtures at sub-attomole levels using this approach.
Lyamichev et al., show that FEN1 nuclease can be used to detect and characterize target DNAs. By adding overlapping pairs of oligonucleotide probes complementary to a predetermined region of target DNA, the cleavage of the downstream probe""s overhanging flap becomes a sensitive indicator of the presence of a target sequence. Multiple copies of the downstream oligonucleotide probe can be cleaved for each target sequence without temperature cycling, which allows amplification of the cleavage signal and also allows quantitative detection of target DNA. By using FEN1 nuclease and fluorescence labeled signal probes, Lyamichev et al. demonstrate that cycling of signal probes can occur when the reaction is carried out at temperatures near the melting temperature of the signal probe. Under these conditions, individual signal probes occupy their complementary site on the DNA target only briefly, allowing for frequent probe exchange (when performed under conditions with excess signal probe) thereby allowing amplification without temperature cycling. Further examples and uses of this approach are described in Dahlberg et al., U.S. Pat. No. 5,888,789 (1999), Kaiser et al., U.S. Pat. No. 5,843,669 (1998) and Dahlberg et al., U.S. Pat. No. 5,837,450. The various nucleases described by Lyamichev, Dahlberg, and Kaiser have been disclosed in Harrington et al., U.S. Pat. No. 5,874,283 (1999) and Dahlberg et al., U.S. Pat. No. 5,614,402.
U.S. Pat. No. 5,637,464 discloses a method for detecting a target oligonucleotide by contacting a sample comprising the oligonucleotide with a labeled primer and an unlabeled helper oligonucleotide which are complementary to the target. Once hybridized, the primer and helper oligonucleotides are joined by DNA ligase.
Both the detection and characterization of specific nucleic acid sequences and sequence changes have been utilized to determine the presence of viral or bacterial nucleic acid sequences indicative of an infection. Further uses of such approaches have allowed for the detection of the presence of variants or alleles of mammalian genes associated with disease, the identification of the source of nucleic acids found in forensic samples, as well as in paternity determinations.
Each of the preceding articles and/or patents describe approaches for detecting and/or characterizing deoxyribo and ribo- nucleic acid molecules. There has been and continues to be a long-felt need for the design of sensitive methods or processes for detecting oligonucleotide compounds, such as antisense therapeutics. Highly sensitive methods would be useful for determining the concentrations of oligonucleotide therapeutics in animal models and/or in the clinic. Further uses would be to study the pharmacokinetic properties of oligonucleotide therapeutics in animal models and/or in the clinic.
The present invention relates to methods and processes for detecting oligonucleotides in bodily fluids and extracts. The methods and processes are particularly useful for quantifying administered modified or unmodified oligonucleotides and/or investigating the pharmacokinetics of a modified or unmodified oligonucleotide compound.
One embodiment of the present invention is a method for detecting or quantitating an oligonucleotide in a bodily fluid or extract, comprising the steps of: contacting the fluid or extract with a probe complementary to the oligonucleotide to form a hybrid, wherein the probe comprises a region at one of its ends which does not hybridize to the oligonucleotide, so that a duplex or hybrid is formed; contacting the hybrid with an enzyme and a detectable label, wherein the enzyme directs the incorporation of the label into the oligonucleotide opposite the region of the probe which does not hybridize to the oligonucleotide; and detecting the label, wherein the presence of the label indicates the presence of the oligonucleotide. Preferably, the body fluid is plasma. Advantageously, the oligonucleotide comprises at least one phosphorothioate linkage. In one aspect of this preferred embodiment, the oligonucleotide comprises a modification at the 2xe2x80x2position of at least one sugar moiety. Preferably, the 2xe2x80x2modification is a 2xe2x80x2-O-methoxyethyl modification. Advantageously, the oligonucleotide comprises at least one modified base. Preferably, the modified base is 5-methylcytosine. In one aspect of this preferred embodiment, the label is calorimetric, radioactive, chemiluminescent, enzymatic or fluorescent. Advantageously, the label is digoxigenin. Preferably, the enzyme is DNA polymerase. In one aspect of this preferred embodiment, the oligonucleotide is exogenously administered.
The present invention also provides a method for detecting or quantitating an oligonucleotide in a bodily fluid or extract, comprising the steps of: contacting the fluid or extract with a capture probe complementary to-the oligonucleotide to form a hybrid, wherein the capture probe comprises a region at one of its ends which does not hybridize to the oligonucleotide; contacting the hybrid with a labeled detection probe complementary to the region of the capture probe which does not hybridize to the oligonucleotide in the presence of an enzyme capable of ligating the oligonucleotide and the detection probe; and detecting the label, wherein the presence of the label indicates the presence of the oligonucleotide. Preferably, the body fluid is plasma. Advantageously, the oligonucleotide comprises at least one phosphorothioate linkage. In one aspect of this preferred embodiment, the oligonucleotide comprises a modification at the 2xe2x80x2 position of at least one sugar moiety. Preferably, the 2xe2x80x2 modification is a 2xe2x80x2-O-methoxyethyl modification. Advantageously, the oligonucleotide comprises at least one modified base. Preferably, the modified base is 5-methylcytosine. In one aspect of this preferred embodiment, the label is calorimetric, radioactive, chemiluminescent, enzymatic or fluorescent. Preferably, the label is digoxigenin. In another aspect of this preferred embodiment, the enzyme is DNA ligase. Advantageously, the oligo nucleotide is exogenously administered.
Another embodiment of the present invention is a method for detecting or quantitating an oligonucleotide in a bodily fluid or extract, comprising the steps of: contacting the fluid or extract with a capture probe complementary to the oligonucleotide and a second probe, wherein the capture probe comprises a detectable marker and a portion which binds to the oligonucleotide, and wherein the second probe comprises a first portion which binds to the detectable marker and a second portion which produces an overhanging flap upon binding to the oligonucleotide to form a complex; contacting the complex with a nuclease to cleave the flap; and detecting the flap. Preferably, the body fluid is plasma. Advantageously, the oligonucleotide comprises at least one phosphorothioate linkage. In one aspect of this preferred embodiment, the oligonucleotide comprises a modification at the 2xe2x80x2 position of at least one sugar moiety. Preferably, the 2xe2x80x2 modification is a 2xe2x80x2-methoxyethyl modification. Advantageously, the oligonucleotide comprises at least one modified base. Preferably, the modified base is 5-methylcytosine. In one aspect of this preferred embodiment, the nuclease is eubacterial polA DNA polymerase, 5xe2x80x2 to 3xe2x80x2 exonuclease, 5xe2x80x2 nuclease associated with bacteriophage T5, FEN1, RAD2 or xeroderma pigmentosum-complementation group G endonuclease homologs from eukaryotes. Preferably, the oligonucleotide is exogenously administered.
Further aspects of the invention are described within the description of the preferred embodiments. The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention and from the claims.